1. Field of the Invention
The present invention relates generally to the fields of bacterial capsule formation and the genes responsible for polysaccharide synthesis. More particularly, it concerns the genes and gene products that direct the formation of the Streptococcus pneumoniae serotype-specific polysaccharide capsule. The present invention also includes the identification of non-type specific gene sequences, flanking the capsule genes, and their use for the directed expression of specific serotypes of S. pneumoniae capsules.
2. Description of the Related Art
Infections due to S. pneumoniae are among the top ten causes of death in the United States. The normal populations most affected are young children and the elderly: pneumococcal pneumoniae, mainly affecting the elderly, causes &gt;40,000 deaths per year among .about.500,000 cases and represents 60 to 80% of all bacterial pneumoniae; pneumococcal meningitis, with .about.4000 cases/year, represents 11% of the total meningitis cases and has a fatality rate of &gt;30%--greater than twice that of the two other leading causes, N. meningitidis and H. influenzae; bacteremia, usually following pneumoniae or meningitis, accounts for &gt;35,000 cases per year (&gt;30% fatal); and otitis media, the most frequent reason for pediatric office visits after well-child care, is caused by S. pneumoniae in .about.50% of cases (ACIP, 1981; ACIP, 1989; Austrian, 1984; Burke et al., 1971; Center for Disease Control, 1978; Johnston and Sell, 1964; Koch and Dennison, 1974).
Other populations have an even higher incidence of pneumococcal infections: approximately 30% of sickle cell children will have severe pneumococcal infections in the first three years of life and .about.35% of those will die (Overturf, et al., 1977; Powars, et al., 1981; Powars, 1975); in both adults and children with HIV infections, S. pneumoniae is the major cause of invasive bacterial respiratory disease (Janoff et al., 1992). Patients with lymphomas, Hodgkins disease, multiple myeloma, splenectomy, and other debilitating diseases or immunologic deficiencies, are particularly susceptible to serious pneumococcal disease, as are those with chronic illnesses such as diabetes mellitus and heart disease. Furthermore, strains of S. pneumoniae are emerging that harbor resistances to multiple antibiotics, including penicillin (Appelbaum, 1992; Jacobs et al., 1978; Landesman et al., 1982).
The polysaccharide capsule of S. pneumoniae is the major virulence determinant of this organism. Despite early studies of the genetics, pathogenesis, and immunology of capsular polysaccharides, it remains unclear why certain capsular types appear to have a greater capacity to cause disease. Of the more than 80 known capsular serotypes, 23 account for more than 90% of all pneumococcal infections.
In children, the most prevalent types are 3, 6, 14, 19, and 23, (Gray and Dillon, 1986), whereas in adults types 1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19, 23 prevail (Finland and Barnes, 1977). In assays of opsonophagocytosis (Branconier and Odeberg, 1982; Giebink et al., 1977; Knecht et al., 1970), complement activation and deposition (Fine, 1975; Gordon et al., 1986; Hostetter, 1986; Stephens et al., 1977; Winkelstein et al., 1980; Winkelstein et al., 1976), and mouse virulence (Briles et al., 1992; Briles et al., 1986; Knecht et al., 1970; MacLeod, 1965; Walter et al ., 1941; Yother et al., 1982), levels of virulence have frequently been found to vary with the type of capsule expressed. For example, isolates expressing type 3, 4, and 19 capsules are highly resistant to phagocytosis, whereas those expressing types 6A, 14, 23 and 37 are significantly less resistant (Branconier and Odeberg, 1982; Hostetter, 1986; Knecht et al., 1970; Wood and Smith, 1949).
The importance of the capsule also results from the fact that anti-capsular antibodies are highly protective against infection. Nonetheless, the current polysaccharide-based vaccine is not particularly useful in some of the populations most affected by pneumococcal disease, e.g., the very young and the elderly, because of poor or absent immune response to polysaccharide antigens.
The ability to produce improved vaccines and therapies for pneumococcal infections will most likely be the result of a better understanding of the basic pathogenic mechanisms of the organism. This understanding necessarily includes the genetic basis for the expression of serotype-specific polysaccharides and the role of capsular type per se in pathogenesis.
Some 85 different serotypes of Streptococcus pneumoniae, differing in the structure of the polysaccharide produced, have been identified (van Dam et al., 1990). The basis for the emergence of new capsule types remains obscure. Whether influenced by mutation, recombination, or immune selection, genetic exchange of DNA is likely to have played a major role in the evolution of capsule types. It is known that pneumococcal capsule types can be changed through genetic transformation in vitro (Dawson, 1930; Dawson and Sia, 1931; Langvad-Nielson, 1944; Avery et al., 1944). Epidemiological studies suggest that a significant degree of genetic exchange occurs in vivo (Crain et al., 1990; Coffey et al., 1991; Versalovic et al., 1993). However, the mechanism by which capsule types are exchanged is not fully understood.
Extensive study was made of the genetics of capsular polysaccharide synthesis in S. pneumoniae using spontaneous mutants with defects in biosynthetic functions (Effrussi-Taylor, 1951; Ravin, 1960; Bernheimer and Wermundsen; 1972). The results of these studies indicated that the genes for polysaccharide synthesis were closely linked and could be transferred as a unit during genetic transformation. A cassette-type model of capsule type change based on this data has been proposed (Taylor, 1949; Austrian et al., 1959; Bernheimer and Wermundsen, 1972). According to the model, the type-specific genes for each capsule type would be present only in the genome of a strain of that capsule type and would show little homology to the type-specific genes of other capsule types. The type-specific genes would be located in homologous sites in the different chromosomes, clustered together between regions of highly homologous flanking DNA. During transformation, recombination would occur in the flanking regions, resulting in the replacement of the recipient's type-specific region by that of the donor.
The clustering of capsule biosynthetic genes proposed by the model is analogous to the organization that has been observed in the gram negative bacteria Escherichia coli (K antigens) (Roberts et al., 1988), Neisseria meningitidis (Frosch et al., 1989), and Haemophilus influenzae (Kroll et al., 1989). For each of these organisms, the type-specific region encoding biosynthetic functions (region 2) is flanked by highly homologous regions necessary for polysaccharide translocation (region 1) and modification (region 3). Since H. influenzae, like S. pneumoniae, is naturally transformable, it has been proposed that capsule type change in this pathogen may occur by transformation with the type-specific gene cluster from a different serotype (Zwahlen et al., 1989).
The one exception to the cassette model of capsule type change in S. pneumoniae is binary capsule formation. When non-encapsulated mutants have been transformed with chromosomal DNA from a strain of a different capsule type, most of the encapsulated transformants express the capsule type of the donor. However, at a frequency 10 to 100 times lower, encapsulated transformants are obtained which express both capsules (Bernheimer and Wermundsen, 1972). In some of these transformants, the second set of capsule genes is closely linked to the original set. However, these strains are unstable, and, at high frequency, lose the ability to produce the original capsule type. In binary strains in which the acquired capsule genes are unlinked to the original genes, binary capsule production is stable (Bernheimer and Wermundsen, 1969). Elucidation of the mechanism of binary capsule type formation may be the key to understanding novel capsule type creation in S. pneumoniae.
It is clear that a better understanding of the genetics of capsular polysaccharide synthesis in Streptococcus pneumoniae is needed. The identification of type-specific capsular genes and the ability to transfer them, singly or as a gene cassette, to desired recipients, will elucidate the role of capsular types in virulence and allow easy identification of S. pneumoniae serotype. This ability will improve existing methods of diagnosis, identifying not only the presence of S. pneumoniae but also the capsular type of the invading strain. Furthermore, it will allow construction of strains producing elevated levels of capsular polysaccharides for improved vaccines.